Pectin Methylesterase and Polyphenol Oxidase

Production of Commercially Suitable

Pectin methylesterase and Polyphenol oxidase

from Agro-industrial Wastes

By

Şebnem ŞİMŞEK

A Dissertation Submitted to the

Graduate School in Partial Fulfillment of the

Requirements for the Degree of

MASTER OF SCIENCE

Department : Food Engineering

Major : Food Engineering

İzmir Institute of Technology

İzmir, Turkey

July, 2004

1

We approve the thesis of Şebnem ŞİMŞEK

Date of Signature

...... 23.07.2004

Assoc. Prof. Dr. Ahmet YEMENİCİOĞLU

Supervisor

Department of Food Engineering

...... 23.07.2004

Prof. Dr. Şebnem HARSA

Department of Food Engineering

...... 23.07.2004

Assist. Prof. Dr. Oğuz BAYRAKTAR

Department of Chemical Engineering

...... 23.07.2004

Prof. Dr. Şebnem HARSA

Head of Food Engineering Department

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ACKNOWLEDGEMENTS

I would like to express my sincere gratitude to my advisor Assoc. Prof. Ahmet Yemenicioğlu for his guidance, supervision, encouragement, and support at all steps of this study.

I am also grateful to my family for their support, encouragement, and understanding.

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ABSTRACT

In this study, some simple and effective extraction and/or partial purification procedures were developed to obtain pectin methylesterase (PME) and polyphenol oxidase (PPO) enzymes from orange peels and mushroom stems, respectively. Also, some characteristics of enzymes were investigated and their stable preparations were obtained in liquid or lyophilized forms. Valencia orange peels contain considerable PME activity (300-350 mL NaOH/min/100 g) that is quite stable during season for at least 5 months. The enzyme was ionically bound to cell walls and can not be extracted by homogenization with water. However, the addition of suitable amounts of NaCl (10 g /100 g extraction mixture) to pellet, obtained by homogenization of peels several times with water, and 30 min mixing (at 200 rpm) may be effectively used to extract the enzyme. The PME in orange peels contains almost the same amount of heat stable and heat labile fractions and the enzyme can not be activated by mild heating. A slight activation (almost 20 %) may be achieved by adding 1 mM CaCl2 to enzyme extracts. However, at higher concentrations the addition of CaCl2 was inhibitory. The PME activity in extracts, stabilized by use of 0.1 % Na-benzoate and 0.1 % K-sorbate, is stable almost 5 months at + 4 oC (maintains > 90 % of its activity). Thus, the commercial preparations of enzyme may be obtained in liquid form. The extracted PME was successfully used to prepare edible films from citrus pectin.

For the extraction of PPO, on the other hand, mushroom stems were first processed to acetone powder. The acetone powders were then extracted with Na-phosphate buffer and partially purified with ammonium sulfate (90 % saturation) or acetone precipitation (2-fold). Following dialysis, the recoveries and purification folds obtained from the partial purification of monophenolase activity of PPO from the same acetone powder were 74-86 % and 3.4-4.3 and 55-67 % and 5.4-6.2 for ammonium sulfate and acetone precipitations, respectively. Thus, it appears that the ammonium sulfate precipitation gives a higher yield but lower purity. The monophenolase activity of partially purified PPO is heat labile and showed inactivation above 45 oC. The enzyme exhibited a pH optimum between pH 6.0 and 8.0. The pH stability of enzyme was maximal at pH 7.0 and 8.0. However, at pH 4.0 the enzyme lost most of its activity after 24 h incubation. The optimum temperature of enzyme was found as 40 oC. The monophenolase activity of PPO enzyme showed no stability in acetone powders at + 4 oC. However, it showed good stability at -18 oC for two months with retention of 60-70 % of its activity. The PPO partially purified with ammonium sulfate precipitation and dialysis, and lyophilized by using dextran or saccharose as supporting materials also retained its monophenolase and diphenolase activities for three months at -18 oC. The effect of lyophilization with dextran on temperature stability of enzyme was insignificant. However, lyophilization with dextran reduced the pH stability of monophenolase activity at 4.0 moderately. In addition to its monophenolase activity on tyrosine and diphenolase activity on L-DOPA, PPO lyophilized with dextran can also use phloridzin as substrate. Thus, it appears that the enzyme may be used in different food applications including the production of antioxidants and colorants, modification of proteins, fermentation of cocoa and black tea, etc.

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ÖZ

Bu çalışmada pektin metilesteraz (PME) ve polifenol oksidaz (PPO) enzimlerinin sırasıyla portakal kabukları ve mantar saplarından eldesi için pratik ve etkili olabilecek ekstraksiyon ve kısmi saflaştırma prosedürleri geliştirilmiştir. Ayrıca enzimlerin bazı karakteristikleri de incelenmiş ve sıvı veya liyofilize haldeki stabil preparatları da hazırlanmıştır. Çalışmada Valencia portakal kabuklarında kayda değer miktarda PME aktivitesi tesbit edilmiş (300-350 mL NaOH/dak/100gr) ve bu aktivitenin sezon boyunca en az 5 ay stabilitesini koruduğu belirlenmiştir. Portakal kabuklarında bulunan enzim, hücre duvarına iyonik olarak bağlı olup su ile ekstrakte edilememekte, ancak buna karşın kabukların su ile birkaç kez homojenizasyonu ve filtrasyonuyla elde edilen kitleden uygun oranda NaCl ilavesi (10 gr/100 gr ekstraksiyon karışımı) ve karıştırma ile (200 rpm’de 30 dak) etkili bir şekilde ekstrakte edilebilmektedir. Portakal kabuklarındaki PME aktivitesinin yaklaşık yarısı ısıya dirençli, yarısı da ısıya duyarlı enzim fraksiyonlarından oluşmakta olup, ısının enzim üzerinde aktive edici bir özelliği belirlenememiştir. Enzim ekstraktına 1 mM CaCl2 ilavesi ile zayıf bir aktivasyon (yaklaşık % 20) sağlanabilmekte, ancak buna karşın yüksek konsantrasyonlardaki CaCl2 ilavesi inhibe edici özellik göstermektedir. % 0.1 Na-benzoat ve % 0.1 K-sorbat ile stabilize edilmiş ekstraktlardaki PME aktivitesi + 4 oC’de yaklaşık 5 ay stabildir (% 90’ın üzerinde aktivitesini korumaktadır). Buna göre portakal kabuğundan elde edilen PME’nin ticari preparatlarının sıvı formda hazırlanmasında herhangi bir sakınca bulunmamaktadır. Bu çalışmada hazırlanmış olan PME preparatı narenciye pektininden yenilebilir film üretiminde başarılı bir şekilde kullanılmıştır.

Diğer yandan, PPO’nun ekstraksiyonu için öncelikle mantar sapları aseton tozuna işlenmiştir. Aseton tozları daha sonra Na-fosfat tamponu ile ekstrakte edilmiş ve % 90 amonyum sülfat çöktürmesi ya da 2–kat aseton çöktürmesi ve bunu takip eden diyaliz işlemi ile kısmi olarak saflaştırılmıştır. Aynı aseton tozundaki PPO’nun monofenolaz aktivitesinin, amonyum sülfat veya asetonla çöktürme yoluyla kısmi saflaştırılması ile elde edilen geri kazanım ve saflık katsayıları sırasıyla % 74-86 ve 3.4-4.3 ve % 55-67 ve 5.4-6.2’dir. Buna göre amonyum sülfat çöktürmesinin daha yüksek verime karşın daha düşük saflık sağladığı görülmektedir. Kısmi olarak saflaştırılmış PPO enziminin monofenolaz aktivitesinin ısıl direnci düşük olup inaktivasyonu 45 oC’ın üzerinde başlamaktadır. Enzim, pH 6.0 ve 8.0 arasında optimum aktivite göstermiş olup pH 7.0 ve 8.0’deki stabilitesi maksimumdur. Ancak buna karşın enzim, pH 4.0’te 24 saat inkübasyon sonucunda aktivitesinin büyük kısmını kaybetmiştir. Enzimin optimum sıcaklığı 40 oC olarak belirlenmiştir. PPO enziminin monofenolaz aktivitesi, aseton tozu halinde + 4 oC’de stabilite göstermemiş, ancak buna karşın - 18 oC’de iki ay süreyle % 60-70 oranında aktivitesini korumuştur. Ayrıca amonyum sülfat ve diyaliz ile kısmi saflaştırılmış ve destek maddesi olarak dekstran ve sakkaroz eklenerek liyofilize edilmiş PPO, –18 oC’de 3 ay süresince monofenolaz ve difenolaz aktivitesini korumuştur. Dekstranla liyofilizasyonun enzimin ısıl stabilitesi üzerinde herhangi bir etkisi belirlenememiştir. Ancak buna karşın dekstranla liyofilizasyon, pH 4.0’deki monofenolaz aktivitesinin stabilitesini kısmen azaltmıştır. Tirozin üzerindeki monofenolaz aktivitesi ve L-DOPA üzerindeki difenolaz aktivitesinin yanısıra dekstranla liyofilize edilmiş PPO, floridzin’i de substrat olarak kullanabilmektedir. Bu sonuç enzimin antioksidan ve renk maddelerinin üretimi, proteinlerin modifikasyonu, kakao ve siyah çayın fermantasyonu gibi farklı gıda uygulamalarında kullanılabileceğini göstermektedir.

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TABLE OF CONTENTS

LIST OF FIGURES...... / xii
LIST OF TABLES...... / xiv
CHAPTER 1. INTRODUCTION...... / 1
CHAPTER 2. ENZYME PRODUCTION FOR INDUSTRIAL APPLICATIONS / 3
2.1. Extraction of Enzymes...... / 3
2.1.1. Cell disruption methods...... / 5
2.1.1.1. Mechanical methods...... / 5
2.1.1.2. Nonmechanical methods...... / 5
2.2. Clarification of Enzyme Extracts...... / 6
2.2.1. Centrifugation...... / 6
2.2.2. Filtration...... / 6
2.2.3. Flocculation and flotation...... / 7
2.3. Concentration of Enzymes...... / 7
2.3.1. Addition of a dry matrix polymer...... / 7
2.3.2. Freeze-drying (Lyophilization)...... / 7
2.3.3. Ultrafiltration...... / 8
2.3.4. Precipitation ...... / 9
2.3.4.1. Precipitation by increasing the ionic strength (salting out)...... / 9
2.3.4.2. Precipitation by decreasing the ionic strength (salting in)...... / 10
2.3.4.3. Precipitation by organic solvents...... / 10
2.3.4.4. Precipitation by alteration of pH...... / 11
2.3.4.5. Precipitation by organic polymers...... / 11
2.3.4.6. Precipitation by denaturation...... / 11
2.3.5. Aqueous two-phase partitioning...... / 12
2.3.6. Removal of salts and exchange of buffers...... / 12
2.3.6.1. Dialysis...... / 12
2.3.6.2. Diafiltration...... / 13
2.3.6.3. Gel filtration...... / 13
2.4. Purification ...... / 13
2.5. Product Formulation...... / 14
CHAPTER 3. PECTIN METHYLESTERASE...... / 16
3.1. Pectin methylesterase and Other Pectinases...... / 16
3.2. Sources of PME...... / 16
3.3. The Effects of PME on Food Quality...... / 17
3.4. Industrial Applications of PME...... / 19
3.4.1. Clarification of fruit juices...... / 19
3.4.2. Firming of fruits and vegetables before processing...... / 21
3.4.3. Modification of pectin...... / 21
3.4.4. Production of low sugar jams, jellies, and compotes...... / 22
3.4.5. Other applications...... / 22
CHAPTER 4. POLYPHENOL OXIDASES...... / 24
4.1. Polyphenol oxidases...... / 24
4.2. Substrates of PPO...... / 26
4.3. Sources and Some Characteristics of PPO...... / 27
4.4. The Effects of PPO on Food Quality...... / 29
4.5. Industrial Applications of PPO...... / 29

4.5.1. Enzymatic cross-linking of proteins or polysaccharides......

/ 30
4.5.2. Production of flavonoid-derived colorants and antioxidants...... / 30
4.5.3. The removal of haze forming polyphenols from beverages...... / 31
4.5.4. Oxygen scavenging and removal of undesirable phenolics from food..... / 32
4.5.5. Removal of undesirable phenolics from wastewaters...... / 32
4.5.6. Analytical and clinical applications of PPO...... / 33
4.5.6.1. Production of biosensors...... / 33
4.5.6.2. Clinical applications...... / 33
CHAPTER 5. MATERIALS AND METHODS...... / 35
5.1. Materials...... / 35
5.2. Methods...... / 35
5.2.1. Methods related to PME enzyme...... / 35
5.2.1.1. PME extraction...... / 35
5.2.1.2. Determination of PME activity...... / 37
5.2.1.3. Effect of mild heating on PME activity...... / 37
5.2.1.4. Effect of CaCl2 on PME activity...... / 38
5.2.1.5. Preparation of a commercial PME preparation and test of its
stability...... / 38
5.2.1.6. Test of obtained PME in the preparation of edible pectin
films...... / 38
5.2.2. Methods related to PPO enzyme...... / 39
5.2.2.1. Acetone powder preparation...... / 39
5.2.2.2. PPO extraction ...... / 39
5.2.2.3. Ammonium sulphate precipitation...... / 39
5.2.2.4. Acetone precipitation...... / 39
5.2.2.5. Determination of PPO activity...... / 40
5.2.2.6. Characterization studies...... / 40
5.2.2.7. Storage stability of PPO in acetone powders...... / 41
5.2.2.8. Preparation of commercial PPO preparations and test of
their storage stabilities ...... / 41
5.2.2.9. The effect of lyophilization with dextran on temperature
and pH stability of PPO...... / 41
5.2.3. Determination of protein content...... / 42
CHAPTER 6. RESULTS AND DISCUSSION...... / 43
6.1. The Results Obtained for PME Enzyme...... / 43
6.1.1. Change of PME activity in orange peels during season...... / 43
6.1.2. Effect of different extraction procedures on PME activity...... / 44
6.1.3. Effect of NaCl concentration on PME activity extracted from
orange peels...... / 45
6.1.4. Effect of extraction period on PME activity extracted from orange
peels...... / 46
6.1.5. Effect of mild heating on PME activity...... / 47
6.1.6. Effect of CaCl2 on PME activity...... / 48
6.1.7. Stability of the prepared PME during storage...... / 49
6.1.8. Test of obtained PME in the preparation of edible pectin films ...... / 50
6.2. The Results Obtained for PPO Enzyme...... / 52
6.2.1. Monophenolase and diphenolase activities of PPO...... / 52
6.2.2. Distribution of PPO in mushrooms ...... / 53
6.2.3.Partial purification of PPO...... / 54
6.2.4. Characterization of monophenolase activity ...... / 56
6.2.5. Stability of monophenolase activity in acetone powders...... / 60
6.2.6. Stability of the prepared PPO during storage...... / 61
6.2.7. The effect of lyophilization with dextran on temperature and pH
stability of PPO...... / 63
6.2.8. The ability of the prepared PPO to oxidize phloridzin...... / 64
CHAPTER 7. CONCLUSIONS...... / 66
REFERENCES...... / 68

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LIST OF FIGURES

Figure 2.1. / The important steps of enzyme production...... / 4
Figure 2.2. / Phase diagram for water...... / 8
Figure 2.3. / Typical profile for ammonium sulphate precipitation...... / 10
Figure 3.1. / Deesterification of pectin by PME...... / 16
Figure 4.1. / The reactions of polyphenol oxidases ...... / 25
Figure 4.2. / The nonenzymatic reactions during formation of dark colored melanins...... / 25
Figure 4.3. / Some of the good substrates of PPO in plants...... / 26
Figure 5.1. / Standart curves for protein determination...... / 42
Figure 6.1. / Change of PME activity in orange peels during season...... / 43
Figure 6.2. / Effect of NaCl concentration on PME activity extracted from peels...... / 46
Figure 6.3. / Effect of extraction period on PME activity extracted from peels...... / 46
Figure 6.4. / Effect of heating for 30 min at different temperatures on PME activity...... / 47
Figure 6.5. / Effect of heating at 50 o C on PME activity...... / 48
Figure 6.6. / Effect of different CaCl2 concentrations on PME activity...... / 49
Figure 6.7. / Stability of the prepared PME during storage at + 4 oC in liquid form...... / 50
Figure 6.8. / The effect of prepared orange peel PME on film forming ability of pectin...... / 51
Figure 6.9. / Monophenolase activity of PPO from mushroom stems...... / 52
Figure 6.10. / Diphenolase activity of PPO from mushroom stems...... / 53
Figure 6.11. / Temperature profiles of monophenolase activity of PPO partially purified with different procedures from different acetone powders / 57
Figure 6.12. / The effect of pH on monophenolase activity of PPO partially purified with different procedures from different acetone powders / 58
Figure 6.13. / pH stabilities of monophenolase activity of PPO partially purified with different procedures from different acetone powders / 59
Figure 6.14. / Optimum temperature for monophenolase activity of PPO...... / 60
Figure 6.15. / The stability of monophenolase activity of PPO in acetone
Powders...... / 60
Figure 6.16. / The stability of monophenolase activity of PPO in different lyophilized forms stored at -18 oC / 62
Figure 6.17. / The stability of diphenolase activity of PPO in different lyophilized forms stored at -18 oC / 62
Figure 6.18. / The effect of lyophilization with dextran on heat stability of monophenolase activity of PPO / 63
Figure 6.19. / The effect of lyophilization with dextran on pH stability of monophenolase activity of PPO / 64
Figure 6.20. / The phloridzin oxidation of PPO lyophilized with dextran...... / 65

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LIST OF TABLES

Table 2.1. / Different methods for cell disruption...... / 5
Table 2.2. / The main chromatographic techniques and their separation principles...... / 14
Table 3.1. / Some properties of PMEs from different fruits and vegetables...... / 18
Table 4.1. / Some properties of PPOs from different fruits and vegetables...... / 28
Table 6.1. / The PME activities obtained by applying different extraction procedures...... / 44
Table 6.2. / Distribution of PPO monophenolase and diphenolase activities in mushrooms...... / 54
Table 6.3. / Results of partial purification with ammonium sulfate and acetone precipitation of PPO monophenolase activity from mushroom stems / 55

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CHAPTER 1

INTRODUCTION

Recently, extensive studies have been conducted related to the use of plant enzymes in food industry. For example, Ridgway et al (1997) extracted and used apple polyphenol oxidase (PPO) successfully for biosynthesis of antioxidant compounds 3-hydroxyphloridzin and 3-hydroxyphloretin from phloridzin and yellow/orange colored dimerized oxidation products of phloridzin that may be used as food colorants. Ridgway and Tucker (1999) also developed a procedure for the partial purification of commercially suitable PPO from apple leaf. However, since PPO in apples can not oxidize the p-diphenols (laccase activity) and lacks monophenolase activity it may only be used for some specific applications.

The monophenolase activity of PPO is essential for many different applications such as the production of plant pigments such as red-violet betalains and gold colored aurones (Strack and Schiliemann 2001) and biosynthesis of antioxidant compounds such as hydroxytyrosol (Espin et al. 2001). Thus, with its hydroxylation and high oxidation capacity PPO in mushrooms is quite suitable for many food applications including fermentation of cocoa and tea (Ridgway et al. 1997, Selamat et al. 2002), removal of undesirable odors caused by volatile sulfur compounds (Negishi et al. 2001, Negishi et al. 2002) and enzymatic cross-linking of proteins (Thalmann and Lötzbeyer, 2002). The ability of mushroom PPO to oxidize sinapic acid was also reported by Choi and Sapers (1994). Thus, as done by Lacki and Duvnjak (1998) with Trametes versicolor PPO, removal of sinapic acid and increase of the nutritional values of canola meal and canola protein concentrates may be conducted by mushroom PPO. One of the most interesting features of mushroom PPO is its ability to inhibit the attachment of some bacteria. For example, Cowan et al (2000) demonstrated that by oxidizing the critical tyrosine residues of glucan binding lectin and glucosyltransferases, PPO prevents the attachment of Streptococcus sobrinus, bacteria responsible from the formation of oral cavities, to glucans deposited on the tooth surface. Kolganova et al (2002) also showed that the PPO reduces the adhesion of some viruses and pathogenic bacteria to buccal epithelial cells, while unaffecting the attachment of probiotic bacteria. These findings are quite interesting and may open the way of using PPO in foods such as gums and confectionaries to increase the tooth health of people. Out of food industry, mushroom PPO may also be used to remove undesirable phenols from wastewaters (Ikehata and Nicell 2000) and produce biosensors for the detection and quantification of phenolic compounds (Rubianes and Rivas, 2000, Climent et al. 2001). Several clinical applications such as using PPO as a catalyst to produce L-DOPA, a drug for the treatment of Parkinson`s disease (Sharma et al. 2003), a marker of vitiligo, an autoimmune disease and a tumor suppressing and prodrug therapy agent (Seo et al. 2003) also attracts considerable interest.

Another enzyme, attracting great attention in food processing is pectin methylesterase (PME). The fungal PME is now extensively used in beverage industry for fruit juice extraction and clarification (Alkorta et al. 1998, Bhat, 2000, Demir et al. 2001, Cemeroğlu and Karadeniz 2001, Kashyap et al. 2001, Sarıoğlu et al. 2001). The use of enzyme in the modification of pectin, oil extraction, firming of fruit and vegetables and peeling of fruits becomes also very popular (Ralet et al. 2001, Schmelter et al. 2002, Degraeve et al. 2003, Suutarinen et al. 2000, Suutarinen et al. 2002, Pszczola, 2001, Kashyap et al. 2001, Vierhuis et al. 2003, Pretel et al. 1997, Janser, 1996). Currently, the plant PME is not used in food industry. However, there are some successful experimental studies related to the use of commercial citrus PME for the firming of fruits and vegetables and modification of pectin (Suutarinen et al. 2000, Massiot et al. 1997).

In the production of commercial enzymes, microorganisms are the primary sources. However, 15 % of the enzyme production is still provided by extracting from animal or plant sources. In this study, the main objectives were; (1) to develop some simple and effective extraction and/or partial purification procedures for PPO and PME enzymes from agro-industrial wastes and (2) to process the extracted enzymes to commercially suitable preparations. The waste materials used in the study were mushroom stems and orange peels for the PPO and PME enzyme, respectively. These, materials are known as rich sources of the indicated enzymes (Moore and Flurkey 1989, Ratcliffe et al. 1994, Seo et al. 2003, Cemeroğlu et al. 2001, Nielsen and Christensen 2002, Johansson et al. 2002, Cameron et al. 1994, Cameron et al. 1998). The enzymes obtained from edible plant materials mostly need no toxicity tests for food applications. Also, because of the non-complex nature of the plant extracts these enzymes may easily be used as crude or partially purified preparations. Moreover, the use of waste materials in enzyme extraction may provide an extra income to factories and reduces the costs of waste material treatments.

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CHAPTER 2

ENZYME PRODUCTION FOR INDUSTRIAL APPLICATIONS

2.1. Extraction of Enzymes

In the production of commercial enzymes, microorganisms are the primary sources. Currently, 50 % of the commercial enzymes are obtained from fungi and yeast and 35 % are obtained from bacteria. The remaining enzyme production, on the other hand, is conducted by extraction from plant or animal sources (Rolle, 1998). A good example for commercial animal origin enzymes is rennet, whereas papain may be an example for the plant origin enzymes (Gölker, 1990).

Following the production of enzymes by microbial fermentation or providing suitable plant or animal sources the first step to obtain industrial enzymes is extraction. In this process, the enzymes are extracted from a source or from a fermentation media and then in the second step the extracts are purified by further processing. According to the mode of application, the degree of purity may range from raw enzymes (sometimes relatively crude preparations in the form of plant executes, chopped fruits, leaves and pounded grains) to highly purified forms. The overall steps for the preparation of enzymes from different sources were given in Figure 2.1.

For the extraction of an enzyme from a plant material (root, stem, grain, nuclear sap, etc.), the material is first ground or minced with different crushers or grinders. At this step, processes such as peeling, removal of seeds, etc. may also be applied. After that, desired enzymes can be extracted with water and/or suitable buffer solutions. On the other hand, when animal organs are used in the extraction, they must be transported and stored at low temperatures for short times to retain the enzymatic activity. Most animal proteins are present in specific muscles or organs surrounded with a fatty layer that often interacts with subsequent purification steps. Thus, before freezing operation fats and connective tissues should be removed. The enzyme containing frozen organ or tissue can be cut, minced or homogenized with a blender, grinder or mill (for hard tissues) for producing a cell paste. Then, the enzyme is extract with an appropriate buffer solution.